Approaches to Improve the Quantitation of Oxytocin in Human Serum by Mass Spectrometry

The neuropeptide oxytocin (OT) regulates several peripheral and central functions and is a molecule of interest in psychiatric diseases such as autism spectrum disorder, schizophrenia, anxiety and depression. The study of OT in human serum samples is however hampered by inconsistent sample preparation and analysis as well as low endogenous blood concentration (1–10 pM). This results in varying reports on OT’s blood levels and interpretation of OT’s role in different (patho)physiological states. Quantitative mass spectrometry (MS) is a highly promising technology to address this problem but still requires large sample volumes to achieve adequate sensitivity and reliability for the quantitation of compounds at low concentrations. We therefore systematically evaluated sample preparation methods for MS to achieve a reliable sample preparation protocol with good peptide recovery, minimal matrix effects and good overall method efficiency in line with FDA guidelines for bioanalytic method development and validation. Additionally, we investigated a strategy to improve the ionization efficiency of OT by adding charged and/or hydrophobic moieties to OT to improve the lower limit of quantitation. Optimized sample preparation in combination with OT modification with a quaternary pyridinium ion improved the sensitivity of OT by ∼40-fold on a tandem triple quadrupole mass spectrometer (API4000 QTRAP), resulting in a lower limit of quantitation of 5 pM in water (linear range 5 pM – 1 mM) and 2 nM in human serum (linear range 2 nM – 1 mM) compared to 200 pM in water and 86 nM in serum with unmodified OT. This approach and protocol provide a solid foundation towards method development for OT quantitation using MS, which should be of high value for fundamental research as well as clinical monitoring of OT upon drug treatments.


Solid phase peptide synthesis of OT analogues with hydrophobic and/or charged residues (for investigation of ionisation efficiency)
OT (H-CYIQNCPLG-NH2) and OT analogues were synthesised on an automated microwave-assisted peptide synthesiser (Liberty Prime, CEM) via Fmoc-SPPS on 0.1 mmol scale on a Rink-amide resin (RAM; RAPP Polymer, 0.74 mmol/g). Fmoc deprotection was performed using 25% pyrrolidine in DMF. Amino acid couplings (5 eq.) were carried out with DIC/Oxyma Pure ® . Fmoc-AA were activated in a mixture of Fmoc-AA-OH/DIC/Oxyma (1:2:1). Upon completion of the peptide chain, the resin was washed with DCM and MeOH. Cleavage from resin and removal of the side-chain protecting groups was achieved by treatment with TFA/TIPS/EDT/H2O (92.5: 2.5: 2.5 : 2.5) at 40°C for 40 min. The resin was filtered off, followed by peptide precipitation with ice-cold Et2O. The peptide suspension was centrifuged, and the peptide pellet was washed 3x with ice-cold Et2O. The Et2O was decanted, and the peptide pellet was dissolved in 50% ACNaq/0.1% TFA and lyophilised overnight. The crude peptide was analysed via LC-MS. OT was oxidised by stirring the linear peptide overnight in 0.1M ammonium bicarbonate buffer (0.1 mg/mL, pH 8.2). For oxidative folding of OT analogues, the linear peptides (0.1 mg/mL) were dissolved in 20-30% ACNaq/0.1% TFA. An iodine solution (10 mg/mL iodine in methanol) was added dropwise during intensive stirring, until the peptide solution turned slightly yellow/brown; stirring was continued stirring for 1 min. The oxidation was stopped by addition of ascorbic acid until the solution turned colourless. The folded peptides were purified by preparative RP-HPLC (Table S1). Cleavage, deprotection, oxidative folding and purification of the peptides was monitored by analytical RP-HPLC and ESI-LC/MS (Table S1).

Quantitation of the peptide concentration to prepare standard solutions
Synthetic peptides contain varying quantities of salts and trapped water molecules from purification/lyophilisation procedures. To determine the actual peptide content and purity, analytical RP-HPLC was performed (Table S1) and compared against two peptide standards with known peptide content established by amino acid analysis (OT, vasopressin). Using the Beer-Lambert Law, the peptide concentrations were calculated based on absorbance of standards and samples using calculated extinction coefficients (Buck et al., 1989, Moffatt et al., 2000, Conibear et al., 2012, Kremsmayr and Muttenthaler, 2022.

LC-MS/MS conditions for sample preparation method development
The LC-MS/MS system consisted of a Shimazu HPLC LC-30 AD binary pump system and tandem quadrupole mass spectrometer (API4000 QTRAP, Sciex) with a TurboIonSpray source employed for detection, which was controlled by Analyst software (version 1.6.2). Chromatography was performed as per conditions listed in Table S2. The API4000 QTRAP was operated in a positive heated ESI mode using multi reaction monitoring (MRM) transition mode. The MRM transition for OT was optimised for m/z 1007.5 (Q1) à 723.0 (Q3) ( Figure S1).
All measurements were carried out on a Shimadzu LC-30 HPLC coupled to a API4000 QTRAP, which was operated in positive electrospray ionisation and multiple reaction monitoring (MRM) mass transition mode. The precursor ion (Q1 ion) for OT was m/z 1007.5 and the product ion (Q3 ion) was m/z 723.0. Fragmentation of Q1 ion broke the peptide bond between Cys 6 and Pro 7 , resulting in a b (Q3 ion) and y-ion ( Figure S1). The b-ion was used for method development due to its higher signal intensity on the API4000 QTRAP compared to the y-ion.

Optimisation of MS analysis method
A 0.1 mM solution of the peptide analyte of interest was prepared and directly injected (15 μL/min) into the API4000 QTRAP to optimise the collision energy (CE) and declustering potential (DP). First, a Q1 scan followed by a product ion scan was performed to identify suitable ions for Q1 and Q3. Then the CE and DP were optimised for each Q1/Q3 transition by running a CE (0-130 V) or DP (0-400 V) ramp (Table S2).

Evaluation of peptide adsorption to glass or plastic HPLC inserts
OT standard solution of 10 μM in H2O, 25%, 50% and 75% ACNaq/0.1% FA were prepared and added to the plastic HPLC inserts (Agilent Technology, item No. 5182-0549) and glass HPLC inserts (SUPELCO, item No. 24707). Each standard was analysed every hour for 24 hours by injecting 1 μL into the API4000 QTRAP. Three independent experiments were carried out. The LC-MS/MS peaks were integrated using Analyst software (version 1.6.2) and all results were expressed as a percentage relative to the peak area of OT in 50% ACNaq/0.1% FA in plastic inserts.

Evaluation of injection volume
A 10 μM OT standard solution in 50% ACNaq/0.1% FA in plastic HPLC inserts was prepared. 1, 5, 10 and 20 μL of OT standard solution were injected (n = 3) into the API4000 QTRAP. Five independent experiments were carried out. LC-MS/MS peaks were integrated using Analyst software (version 1.6.2).

Calibration curve and determining the LLOQ of mass spectrometers
Dilution series of 14 OT standards ranging from 1 mM to 0.1 fM in 50% ACNaq/0.1% FA were prepared and 10 μL of each standard solution injected to the LC-MS/MS instrument for analysis. Standard curves were prepared in three independent experiments and analysed in triplicates. LC-MS/MS peaks were integrated using Analyst software (version 1.6.2). For the standard curve, the logarithmic (log10) peak areas were plotted against the logarithmic (log10) OT concentrations. The linear range was determined by the maximum number of points that could be included for the R 2 coefficient to remain ≥0.9. LLOQ was determined by visual evaluation of the standard curve (smallest value on the linear range, before the plateau) and had to be ≥ 5x of the blank signal). HPLC conditions: 0-90% B in 3 min at 0.25 mL/min (A: H2O/0.1% FA; B: ACN/0.1% FA). HPLC column: Agilent Technologies, Hypersil Gold C18, 100 x 2.1 mm, 3 μm. MS settings were chosen according to tuning results (Table S2).

Evaluation of sample preparation protocols using matrix effect, peptide recovery and overall method efficiency
Six sample preparation protocols were evaluated towards matrix effect (MX), peptide recovery (RE) and overall method efficiency (ME). 100 µL human serum samples were spiked with 10 µL OT standard solutions at three concentrations (0.1 µM, 10 µM and 50 µM), yielding final OT serum concentrations of 4.5 µM, 0.91 µM and 9.09 nM, respectively. Protocols 1 to 3 were performed on a Sep-Pak (50 mg) cartridge (item No. WAT054955) from Waters and on an Oasis HLB (60 mg) cartridge (item No. WAT094226) from Waters, while Protocols 4, 5 and 6 were only tested on Oasis HLB cartridges. MX, RE and ME were determined via the quantitative pre-and post-spike method (Matuszewski et al., 2003). Each protocol was performed on three samples, and each sample was analysed in triplicates. HPLC conditions: 0-90% B in 3 min at 0.25 mL/min (A: H2O/0.1% FA; B: ACN/0.1% FA). HPLC column: Agilent Technologies, Hypersil Gold C18, 100 x 2.1 mm, 3 μM. MS settings were chosen according to tuning results (Table S2).

Evaluation of reconstitution volume and LLOQ of sample preparation Protocol 6'
To determine the MX, RE and ME of each reconstitution volume, 100 μL human serum samples were spiked with 10 µL of OT standard solution (50 µM) and sampled according to Protocol 6' ( Table 3).
Reconstitution volumes of 40, 60, 80 or 100 μL of 50% ACNaq/0.1% FA were analysed. MX, RE and ME were determined via the quantitative pre-and post-spike method. 10 μL of each reconstituted sample was analysed via LC-MS/MS and the chromatographic peaks were integrated using Analyst software (version 1.6.2). To determine the effect of reconstitution volume on LLOQ, 100 µL of human serum samples were spiked with 10 μL of OT standard solutions ranging from 0.1 pM to 1 mM, sampled according to Protocol 6' and reconstituted in 40, 60, 80 or 100 µL of 50% ACNaq/0.1% FA. 10 µL of each sample was analysed by the API4000 QTRAP. The LLOQ of each reconstitution volume was calculated as per Section 2.7.

Evaluation of intraday accuracy and precision of Protocol 6'
A dilution series of 11 OT standards (0.1 pM to 1 mM) were prepared and analysed by the API4000 QTRAP to produce an OT standard curve (n=5). This standard curve was used to calculate the concentration of OT samples prepared with Protocol 6'. 100 µL human serum samples were spiked with 10 µL OT standards (final human serum concentration: 1.5 µM, 10 µM and 40 µM) and purified with Protocol 6'. Samples were reconstituted with 60 μL 50% ACNaq/0.1% FA, and 10 μL of each reconstituted sample was analysed via LC-MS/MS. Chromatographic peaks were integrated using Analyst software (version 1.6.2). Precision was expressed as a percentage of the standard deviation from the mean value of the five experiments, and accuracy as a percentage of the error from the expected value (Administration, 2018).

Evaluation of LLOQ of OT across different mass spectrometers
A dilution series of OT ranging from 1 fM to 0.1 mM in 50 % ACN, 0.1 % FA was analysed on different mass spectrometers, including four quadrupole time-of-flight (QTOF) mass spectrometers (TripleTOF 6600, TripleTOF 5600, X500R, QstarElite) and two triple quadrupole instruments (API4000 QTRAP, QTRAP6500) ( Figure S4). All mass spectrometers were operated in positive ESI-mode. The sensitivity of each instrument was determined by calculating the LLOQ of OT as per Section 2.7.

Evaluation of OT analogue hydrophobicity
The purity and relative hydrophobicity of the synthesised peptides was analysed via UV at 214 nm on a RP-HPLC system (column: Hypersil Gold, 100 x 2.1 mm, 3 μM; method: 0-50 % solvent B in 50 min, 0.3 mL/min). 10 μL of the peptide stock solution (3 mg/mL in 50% ACN, 0.1 % FA) were injected. The data were collected and processed using Shimadzu-Lab solution software (version 5.97). The retention time of each peptide was normalised to OT by subtracting OT`s retention time from the peptide's retention time.

Alkylation of the thiol groups of reduced OT
Reduced OT and 2 eq. of ligands i, ii, iii and iv were mixed in 30% aqueous NaOH solution and incubated at 25°C for 15 min. The reaction was stopped by adjusting the pH to 3 with TFA. The reaction mixture was diluted with H2O/0.05 % TFA and purified with preparative RP-HPLC using a linear gradient 0-50% B (solvent A: H2O/0.05% TFA; B: 90% ACNaq/0.043% TFA) in 100 min at 16 mL/min while monitoring UV absorbance at 214 nm.       Table S7: Conversion of standard concentrations. The molecular weight of OT is 1007.19 g/mol. For simplicity, 1000 g/mol was used for conversions.